Toxoplasma gondii is an obligate intracellular apicomplexan parasite causing severe disease in people with a compromised (by drug or diseases) or immature (congenital infection) immune response. Disease pathogenesis is primarily the result of an uncontrolled expansion of parasite biomass, the associated significant tissue destruction, and ensuing inflammation. The speed of replication (i.e. the length of the cell cycle) is associated with increased virulence. Also, the efficiency of gliding motility correlates with the efficiency to passage biological barriers such as the placenta, and also defines the invasion efficiency of the host cells. Furthermore, enhanced extracellular survival correlates with increased virulence as well. Although these features have been known for some time, we only have a limited grasp on the molecular basis of these virulence factors. Under this proposal we wish to apply a new approach toward elucidating these processes at the molecular level. Our approach is based on the observation that parasite strains maintained in the laboratory for extended periods display features associated with high virulence (fast replication, prolonged extracellular survival, more motile). In the past we, and others, have attempted to correlate these changes with changes in genotype or gene expression. However, these studies have not provided a clear picture, as the contribution of individual genes to the phenotype appears minor. It is our hypothesis that these virulence features originate in the concerted action of the combination of mutations and that taking individual mutations outside their genetic context only has minor effects on the phenotype (i.e. complex and multi-allelic). Therefore, a good understanding of these phenotypes is only possible when considering the complete set of mutations. To overcome this challenge we propose an in vitro experimental evolution experiment starting with a recent parasite isolate into a lab-adapted strain. In vitro evolution is an established approach in protein biochemistry as well as in studies of viral and bacterial virulence, but has not been applied to study parasite virulence. We will select parasites for various specific features associated with virulence, such as prolonged extracellular survival, the efficiency to cross biological barriers and increased replication rate. By comparing the development of the genotype and gene expression profiles with the phenotype throughout the evolutionary process we will be able to unravel the genetic basis of these virulence factors. This will also aid our understanding toward how different genes are functionally correlated, information that is currently completely missing. Making complex and multi-allelic associations was not possible in the previous studies directly comparing recent wild isolates with lab-adapted strains. We therefore expect to significantly enhance our understanding of the various pathways underlying lab adaptation, and by extrapolation, virulence.